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Patients with haematological malignancies are at increased risk of severe disease and death from COVID-19 and are less likely to mount humoral immune responses to COVID-19 vaccination, with the B cell malignancies a particularly high-risk group.Our COV-VACC study is evaluating the immune response to COVID-19 vaccination in patients with B cell malignancies. Eligible patients were either receiving active treatment or had received treatment within the last 24 months. Patients were vaccinated with either the BNT162b2 (Pfizer-BioNTech) (n=41) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca) (n=14) vaccines. The median age of participants was 60 years (range: 27-82) and 50% were receiving systemic anti-cancer therapy (SACT) at the time of vaccination. This interim analysis from the first 55 participants describes anti-S seropositivity rates, neutralising antibody activity and association with peripheral lymphocyte subsets.After the first vaccine dose, 36% overall had detectable anti-S antibodies rising to 42% after the second dose. Sera from seropositive patients was assessed for neutralisation activity in vitro. Of the seropositive patients after first dose (n=17), only 41% were able to neutralise SARS-CoV-2 pseudotyped virus with a 50% inhibitory dilution factor (ID50) of >1:50. After two doses (n=21) 57% of the seropositive patients had detectable neutralisation activity (median ID50 of 1:469, range 1:70 – 1:3056). Total blood lymphocyte, CD19, CD4 and CD56 counts were significantly associated with seropositivity. Patients vaccinated more than 6 months after completing therapy were significantly more likely to develop antibodies than those within 6 months of treatment or on active treatment; OR: 5.93 (1.29 – 27.28).Our data has important implications for patients with B cell malignancies as we demonstrate a disconnect between anti-S seropositivity and virus neutralisation in vitro following vaccination against COVID-19.Urgent consideration should be given to revaccinating patients with B-cell malignancies after completion of anti-cancer treatment as large numbers currently remain at high risk of infection with the increasing transmission of SARS-CoV-2 in many countries.

The optimal treatment for patients with newly diagnosed acute myeloid leukaemia (AML) who are infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2)/COVID‐19 is unknown.1 We report the case of a previously fit 27‐year‐old male who presented with a 3‐day history of fever (>39 C), swollen, erythematous elbows and no respiratory symptoms. His white blood count (WBC) was 187×109/L and bone marrow (Figure 1A & 1C) examination revealed normal karyotype AML with a fms related receptor tyrosine kinase 3 (FLT3) internal tandem duplication (ITD), wild‐type NPM1 and no additional mutations on a next‐generation sequencing panel.

The impact of Coronavirus disease 2019 (COVID-19) on outcomes in patients with cancer remains unclear. Acute Myeloid Leukemia (AML)/high-risk myelodysplasia (MDS) are common hematological malignancies resulting in profound immunosuppression, which is exacerbated by intensive and less-intensive chemotherapy. Importantly, venetoclax based regimens have been increasingly used during the pandemic as a strategy to reduce patient hospitalization however, there is little information concerning the impact of such regimens on COVID-19 infection rates. We therefore opened a prospective clinical study (PACE), at the start of the current pandemic in April 2020 to characterize the risk of COVID-19 infection in patients with AML/MDS-EB2 receiving intensive or non-intensive treatment, including patients treated with venetoclax-based regimens. The primary aim was to determine the incidence of COVID-19 in patients with AML /MDS-EB2 including both, prior to study entry and during treatment until 4 weeks after the last cycle of treatment. Secondary aims were to: characterize the presentation of COVID-19; define the severity and type of both non-COVID-19 and COVID-19 infections; and undertake an exploratory analysis to quantify the incidence of COVID-19 infection in patients receiving (less-intensive) venetoclax based regimens. All analysis conducted to date has been descriptive. 211/230 recruited patients had full treatment histories available, of whom 116 patients received intensive chemotherapy and 95 low intensity regimens. 48 patients received a venetoclax-based regimen. The median age of the non-intensive treatment arm was 72 years; (range 19.1-86.5) and of the intensive arm was 59 years (range 16.1-76.1). There were more cases of secondary AML and relapsed disease in the non-intensive arm as compared to the intensive arm. 25/226 evaluable patients tested positive for COVID-19 as defined by positive SARS-CoV2 PCR test, 10 with a prior diagnosis at study entry and 15 tested positive during the study. The incidence of COVID-19 infection for patients with AML/MDS-EB2 was 11.1% (90%CI: 7.8%-15.1%) (Table). A lower proportion of patients (n=6/91 6.6%) undergoing non-intensive treatment suffered COVID-19 as compared to those undergoing more intensive chemotherapy regimens (n=19/116, 16.4%). Specifically, only 3/48 (6.3%) patients undergoing a venetoclax regimen were infected with SARS-CoV2. The most common presenting symptoms of COVID-19 in this study, regardless of the intensity of chemotherapy, was fever and cough with 6/25 patients asymptomatic. The risk of death at 30 days following study entry in patients who had prior COVID-19 infection or who contracted COVID-19 during this period was 13.6%, compared to 3.9% in the overall cohort without COVID-19 infection. There was a lower incidence of non-COVID-19 related infections in patients receiving venetoclax-based regimens, n=43 infections in 24 (50.0%) of patients; with 313 infections in 94 (81%) of intensively treated patients. The overall occurrence of non-COVID-19 infection in the non-intensive arm was 87 infections in 50 (54.9%) patients. Our multi-center study provides real-world estimates for the incidence and presentation of COVID-19 infection in a cohort of patients with AML/MDS-EB2, and indicates a higher risk of death at 30 days in patients with prior COVID-19 infection prior to, or during treatment. Venetoclax based, and other non-intensive, regimens, increasingly implemented during the pandemic, to minimize patient exposure and reduce usage of hospital beds, appeared to be associated with a low incidence of COVID-19. Further follow-up will be required to understand the long-term impact of this strategy. Analysis of immune responses to COVID-19 infection and vaccination is on-going. Acknowledgments: This study was funded by Cure Leukaemia under the Trials Acceleration Program (TAP), and grants from BMS and Blood Cancer UK. Figure 1 Figure 1. Disclosures Loke: Novartis: Other: Travel; Janssen: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Daichi Sankyo: Other: Travel. Knapper: Pfizer: Consultancy, Speakers Bureau; Astellas: Ended employment in the past 24 months, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Khan: Abbvie: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Jazz: Honoraria; Gilead: Honoraria; Novartis: Honoraria. Dillon: Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Jazz: Other: Education events; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees. Culligan: AbbVie Ltd: Honoraria, Speakers Bureau; Celgene Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Jazz Pharma: Honoraria, Speakers Bureau; Takeda UK Ltd: Honoraria, Speakers Bureau. McMullin: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: clinical trial support, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan: Research Funding, Speakers Bureau. Murthy: Abbvie: Other: support to attend educational conferences.. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction The molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients. Method We searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels. Results 37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%. Discussion The median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 & BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 & EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified. The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling. 1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-679 2. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 Figure 1 Figure 1. Disclosures Sekhar: Novartis: Consultancy, Research Funding.

The outcomes for follicular lymphoma (FL) have improved significantly in recent years. This has been driven by an improved understanding of the pathobiology of FL and the development of therapeutic anti-CD20 antibodies. Combining rituximab with chemotherapy, coupled with its use as maintenance therapy, has contributed to significant improvements in disease control and progression-free survival. However, FL remains incurable and almost all patients invariably relapse. Therefore, there remains a need to develop novel therapeutic options and optimize existing regimens. Obinutuzumab (a first-in-class, glycoengineered, humanized type 2 anti-CD20 antibody) has been evaluated in a number of clinical trials. In this review, we will summarize the evaluable results of clinical trials investigating the efficacy of obinutuzumab in the treatment of FL.

Purpose To assess the risks of second malignancy and early menopause in a large cohort of patients with gestational trophoblastic tumor who were treated with chemotherapy. Patients and Methods A survey of patients treated at Charing Cross Hospital between 1958 and 2000 was performed in 2006 to assemble incidence data for subsequent malignancies and the age at menopause. Treatment records were reviewed for the regimens and durations, and the incidence of subsequent malignancies was compared with that in the national age-matched population. Results Data were obtained for 1,903 patients, with a mean follow-up of 16.9 years. Eighty-six patients developed a subsequent malignancy compared with an expected number of 79 (standardized incidence ratio [SIR], 1.1; 95% CI, 0.9 to 1.3). The overall risk was low for patients treated with single-agent methotrexate and folinic acid (MTX-FA; SIR, 0.7; 95% CI, 0.5 to 1.1) and also for patients treated with etoposide, methotrexate, and dactinomycin followed by cyclophosphamide and vincristine on alternating weeks (EMA-CO) with an SIR of 0.9 (95% CI, 0.4 to 2.2), but there were significantly increased risks of oral cancer, melanoma, meningioma, and leukemia. The cumulative risk of early menopause was low after MTX-FA but was substantial after EMA-CO, reaching 13% by age 40 years and 36% by age 45 years. Conclusion Subsequent cancer risks for patients cured of gestational trophoblastic tumors with modern chemotherapy appear similar to those of the normal population with no overall increased risk of malignancy after MTX-FA or EMA-CO. However, there was evidence of an increased risk of leukemia after EMA-CO and some evidence of other site-specific increased risks based on small patient numbers. All major treatments except MTX-FA increased the risk of early menopause.

Introduction: The optimal management of relapsed/refractory lymphoma is a significant clinical challenge. Early phase clinical trials are primarily designed to assess safety but also represent options for patients with limited therapeutic choices. Outcomes for lymphoma patients on early phase trials have previously been reported as single centre cohorts or grouped analyses with other malignancies. We performed a novel meta-analysis of publically available reports of early phase trials in lymphoma and compared the outcomes with those from our early phase trials unit. Methods: The outcomes of lymphoma patients enrolled on early phase trials at a UK tertiary centre were reviewed. AEs were graded according to CTCAE v4.0 and response criteria evaluated per protocol. Patient and therapy characteristics, AEs and best clinical responses were summarised by descriptive statistics. Individual-patient survival data were analysed using Kaplan-Meier method and survival curves compared with the log-rank test. A systematic literature review was performed using EMBASE, MEDLINE and clinicaltrials.gov to identify publicly available reports of early phase clinical trials reported in 2016-2017 and data was extracted by two independent reviewers. Meta-analyses of ORRs were performed using random-effect models. Results: 50 patients were enrolled onto 9 Phase I and I/II trials between March 2012 and June 2018, Four patients participated in 2 trials, considered separate events. 5 IMPs were small molecular inhibitors, 4 immunotherapies, 4 first in human and 8 investigated as monotherapy. Diagnoses included 42 aggressive NHL (aNHL) [30 DLBCL, 3 PMBCL, 3 Richters, 1 MCL, 5 T cell], 10 indolent NHL (5 WM, 2 FL, 2 MZL, 1 CLL/SLL) and 2 HL. Median age was 54 yr (27-83), 72% male, with a median time from diagnosis of 22.5 months and median 3 prior lines of therapy (range 1-8). Patients received a median number of 2 cycles of IMP (range 1-28) over 57.5 days (IQR: 37-116). 42.6% experienced grade 3-4 toxicity and 31.5% required dose interruptions of >7 days. ORR and clinical benefit rate (≥SD) were 28% and 47% respectively (CR 4%, PR 24%, SD 19%). Patients were followed up for a median of 11.4 months. Median PFS and OS were 2.3 and 6.8 months respectively, with PFS and OS at 3, 6 and 12 months being 45.8%, 34.4%, 26.5% and 58.4%, 45.4% and 38.8%. Median OS was greater for those who received 164 lymphoma trial reports were included in the meta-analysis detailing outcomes of 4537 patients (Table 2). All studies were Phase I (72.6%) or I/II and 78% included only patients with lymphoma (all other trials included reported subgroup analysis of lymphoma patients). 95.7% of trials evaluated a single IMP, 52.4% used combinations of agents. IMPs most frequently investigated were small molecule inhibitors (25.6%), antibody-drug conjugates (11.6%) and epigenetic modifiers (10.4%). Immunotherapy trials comprise 36.1% of studies, including ADCs, checkpoint inhibitors (7.32%), naked antibodies (9.2%) and cellular therapies including CAR-T (7.93%). The ORR of all patients was 54.2% (95% CI 49.6% - 58.8%). Subset analysis showed that cellular therapies studies reported a pooled ORR of 62.5% (50.9 - 72.8) and antibody therapies 58.3 (46.7 - 69.2). Conclusion: The outcomes of lymphoma patients on early phase trials is historically perceived as very poor, partly due to the grouping of analysis with other malignancies. Our cohort had an ORR of 28% and OS at 6 months of 58.4%. The meta-analysis of global studies reporting lymphoma specific outcomes, revealed an ORR of 54.2%. This included all histological subtypes and some previously untreated patients. Our cohort was enriched for relapsed aNHL, which may account for the inferior ORR in our cohort. Together, both data sets indicate improved outcomes compared to historical reports and support enrolment of suitable patients into phase I trials when conventional options are exhausted. Disclosures Ardeshna: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Takeda: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Popat:Amgen: Honoraria. Townsend:Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria.

Introduction:The overall survival (OS) for patients with multiple myeloma (MM) continues to improve with advances in therapies such as second-generation proteasome inhibitors (PI), immunomodulatory agents (IMiDs) and monoclonal antibodies. Advanced therapeutics (eg chimeric antigen receptor (CAR-T) cells), immunotherapies and 3rd generation agents are also under evaluation. Therefore, interest in Phase 1 clinical trials has increased as patients may benefit from exposure to a novel therapy. Also, increasingly data has been used to obtain breakthrough designation from regulatory authorities as part of accelerated approvals. However, these therapies have unproven efficacy, may cause potentially severe toxicities and patients may receive sub-optimal doses. We therefore analysed the outcomes of our patients and to complement this performed a meta-analysis of publicly available Phase I MM trials. Methods: This was a retrospective review of patients with MM enrolled onto phase I and I/II trials at the NIHR/UCLH Clinical Research Facility, London, UK. Patients received at least one dose of study drug, AEs were graded by CTCAE 4.0 and response criteria evaluated as per protocol. Individual-patient survival data were analysed using the Kaplan-Meier method and survival curves compared with the log-rank test. A meta-analysis using Medline, EMBASE and clinicaltrials.gov was performed to identify abstracts/papers reporting phase I trials from January 2016 to December 2017. Data was extracted by two independent reviewers using the same inclusion and exclusion criteria with significant overlap to ensure inter-observer consistency. Meta-analyses of Overall Response Rate (ORR) were performed using random-effect models. Results: 50 patients were enrolled onto 7 phase I or I/II clinical trials between March 2012 to June 2018 at the NIHR/UCLH Clinical Research Facility. 6 participated in 2 trials (each trial entry considered a separate event). IMPs were: monoclonal antibody (Mab) (1), HDAC inhibitor (1), immune-modulator (1), proteasome pathway inhibitors (2), kinase pathway inhibitor (1), epigenetic modifier (1). 3 trials were monotherapy and 4 as combinations. The median age was 54 years (33-73), 46% were female. The median time from diagnosis to trial entry was 58.5m (IQR 36-104.5) and median prior lines of therapy was 3 (0-7). Patients received a median of 10 cycles (1-34) over a median duration of 176 days (IQR 112-425). Study withdrawal was due to progressive disease (50%), autograft (16.1%), toxicity (12.5%), completion of study (12.5%) and other (3.6%) (Table 1). 30.4% of patients experienced G3-4 toxicity and 28.6% had dose interruptions of ≥7 days. The overall response rate (≥PR) was 55.3%, ≥VGPR: 26.7% (see Table 1).The median PFS was 14.2 m (95% CI:7.6-15.9) and OS 37.3m (95% CI:29.2-not reached) with a median follow-up of 26.5m. Median OS for those with < 4 prior lines was better (48.1m) vs those ≥ 4 prior lines (30.6m, p-value log-rank test=0.03). Those with adverse risk cytogenetics appeared to do worse vs standard risk: PFS 14.2 vs 7.6m (log rank not significant); OS 37.3 vs 30.6m (log rank: not significant). Most patients (80.3%) received further treatment after trial (12.5% received another trial, 46.4% chemotherapy; 21.4%: autograft). 8.9% were managed expectantly and 5.4% palliated. 110 trial abstracts & publications were selected for meta-analysis that included 3222 patients with a median age of 64 years (30-75), 42% female, median 4 prior lines. 64.6% of reports were combination studies. 17% targeted the proteasome, 17% were MAbs, 10.9% were epigenetic modifiers, 5.5% were cellular therapies and 24.6% were other small molecule inhibitors (Table 2). The overall response rate across all studies was 52.2% (95% CI 46-58.2). Higher responses were described for immunotherapy studies: BCMA CAR-T cells, 74.2% (95%CI 42.3- 91.8) and monoclonal antibodies, 64.2% (95% CI 56.2- 71.4). PFS and OS was not consistently reported across studies. Conclusions: Patients derive benefit from phase 1 trials with clinically significant responses reported from this single centre and meta-analysis. Additionally, most received further treatment after. The best responses were reported for immunotherapy studies. Despite this, in our data there was an overall trend for worse outcomes for adverse risk cytogenetics and for those treated later in their disease course. Disclosures Townsend: Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Yong:Takeda: Speakers Bureau; Amgen: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau. Popat:Amgen: Honoraria.

The Polo-like kinases (Plks) are a highly conserved family of protein kinases that function in regulation of cell cycle and DNA damage-induced checkpoints. Evidence of a tumor suppressor function for the Plks in human neoplasia is lacking. Here, we report that Snk/Plk2 is transcriptionally down-regulated in B-cell neoplasms. Silencing occurs with very high frequency in Burkitt lymphoma (BL) but is also detected in B-cell neoplasms of other types and is associated with aberrant cytosine methylation in the CpG island located at the 5' end of the SNK/PLK2 gene. Silencing is specific to malignant B cells because SNK/PLK2 was unmethylated (and expressed) in primary B lymphocytes, in EBV-immortalized B lymphoblastoid cell lines (LCLs), and in adenocarcinomas (of the breast) and squamous-cell carcinomas (of the head and neck). Expression of Snk/Plk2 in BL cell lines was restored by demethylating agents. The related PLK1 and PLK3 (FNK/PRK) genes were overexpressed in BL cell lines lacking Snk/Plk2 expression, consistent with functional degeneracy among the Plk family. Ectopic expression of Snk/Plk2 in BL cells resulted in apoptosis, a potential mechanistic basis underlying the strong selective pressure for abrogation of Snk/Plk2 function in B-cell neoplasia.

A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein–Barr virus (EBV)-encoded nuclear protein and the C8 (α7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/α7. The interaction between these viral proteins and GST–C8/α7 was shown to be significantly more robust than the previously reported interaction between C8/α7 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Co-immunoprecipitation of the EBNA3 proteins with C8/α7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an α-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.

Human B cells isolated from peripheral blood were activated and induced to proliferate by either Epstein–Barr virus (EBV) or the T cell-derived mitogens CD40 ligand (CD40L) plus interleukin (IL)-4. Although both populations initially proliferated as B-blasts, significant differences were revealed over a longer period. EBV infection resulted in continuously proliferating lymphoblastoid cell lines (LCLs), whereas most of the CD40L/IL-4-stimulated B cells had a finite proliferative lifespan of 3–4 weeks. Cell cycle analysis, trypan blue staining and Western blot analysis for cleavage of poly(ADP-ribose) polymerase (PARP) all demonstrated that the decrease in proliferation in CD40L/IL-4-stimulated B cells is not due to cell death. Instead, these cells arrest, accumulate in G0/G1and undergo alterations in cell surface marker expression, cellular morphology and immunoglobulin production, all consistent with plasmacytoid differentiation. In contrast, B cells infected with EBV continued to proliferate and retained a blast-like phenotype. Differences in both cytokine production and the expression of cell cycle regulators were identified between the two B-cell populations, which might contribute to the differentiation of the CD40L/IL-4-stimulated B cells and suggest potential mechanisms by which EBV may overcome this. The study has also identified a window of opportunity during which a comparison of isogenic populations of EBV- and mitogen-driven B blasts can be made.

Upregulation of p53 protein induces either growth arrest or apoptosis in response to cellular injury This is signaled from a highly conserved p53 domain between codons 64 and 92, where a functional polymorphism results in either a proline (p53-72P) or an arginine (p53-72R) at codon 72. Preliminary studies suggest that p53-72R may be a risk factor for cervical cancer and, consistent with this, preferential mutation and retention of the p53-72R allele has also been demonstrated in other cancers of squamous cell origin. Here we examine the relationship between allelic forms of p53 and nonmelanoma skin cancer, by determining the correlation with susceptibility to sunburn, which is a known risk factor, and then by p53 sequence analysis of a large series of tumors. We found a significant positive association between p53-72R and susceptibility to sunburn, as assessed by skin phototype and minimal erythemal dose following solar-simulated radiation (p = 0.0001 for trend). We also found a significant association between p53-72R homozygosity and nonmelanoma skin cancer in renal transplant recipients (basal cell carcinoma, p < 0.01; squamous cell carcinoma, p < 0.05) but not in immunocompetent patients compared with skin type matched controls. p53 sequence data revealed mutations in 30 of 70 (42.9%) nonmelanoma skin cancers, 28 (93%) of which were in the p53-72R allele. Loss of heterozygosity occurred more frequently in p53-72RP than in p53-72RR tumors (p = 0.0001) with preferential loss of p53-72P in heterozygotes (p = 0.016), irrespective of the mutant status of the concomitant allele. Together these data infer functional differences between polymorphic forms of p53 that are likely to be relevant to skin carcinogenesis.

Undifferentiated nasopharyngeal carcinoma (NPC) is an epithelial malignancy that is consistently associated with Epstein-Barr virus (EBV) but which very rarely has p53 gene mutations in primary tumours. Since the tumour suppressor p53 is mutated in most human cancers or the wild type protein is inactivated in a significant number of the remainder, here we have investigated cellular factors that could compromise p53 function in primary NPC. Twenty-five primary tumours were judged to carry only wild type p53 by SSCP analysis of all exons and sequence determination of exons 4-9. Only one tumour was found to express significant levels of hMdm2 and in 24/25 there were no detectable mutations or deletions in exons 1beta and 2 of the p14(ARF) gene. However, immunohistochemistry consistently revealed that all the tumour cells express substantial amounts of the p53-related protein p63. Semi-quantitative RT-PCR analysis of mRNA from tumour biopsies showed that the dominant species expressed was invariably the truncated deltaN-isotype. Since this can block p53-mediated transactivation, it is potentially a dominant-negative isoform. In normal nasopharyngeal epithelium the distribution of p63 was restricted to the proliferating basal and suprabasal layers. We suggest that deltaN-p63 is a good candidate as a suppressor of wild type p53 function in these tumours and also that it may prove to be a valuable diagnostic marker for undifferentiated NPC.

To determine whether latent Epstein-Barr virus (EBV) modifies DNA damage responses in B lymphocytes, cells were treated with agents either producing DNA cross-links and adducts or generating double-strand breaks. The cyclin-dependent kinase inhibitor p21 WAF1 accumulated in mitogen-stimulated primary B cells following exposure to all genotoxins tested. In contrast, when proliferation was EBV driven, p21 WAF1 failed to accumulate after treatment with the DNA adduct-producing agents. The tumor suppressor p53 was stabilized and phosphorylated after all treatments, irrespective of whether latent EBV was present. This suggests that regulatory pathways upstream of p53 are unaffected by latent EBV but downstream effectors are altered if DNA adducts or distortions are involved.

Publisher Summary This chapter reviews a considerable body of evidence, which indicates that Epstein–Barr Virus (EBV) can disrupt the regulation of the cell cycle. Although EBV can not appear to directly target the major tumor suppressors p53 and pRb, it encodes proteins—that when expressed during the latency III proliferation programme—can modulate activities of key proteins in checkpoint control. Although EBV differs from the other human tumor viruses. It is now generally accepted that oncoproteins encoded by tumor viruses can drive genomic instability, and this is best characterized for the human papillomavirus proteins E6 and E7. Here genomic aberrations result from a general reprogramming of the cell cycle machinery, but particularly the deregulation of cdk2. There are now clear indications that EBV disrupts the same regulatory networks, but that this might not occur via precisely the same molecular interactions. Therefore—although details of the molecular interactions need to be established—a new conceptual framework is emerging that hopefully can lead to a fuller understanding of EBV-associated B-cell transformation and B-cell cancer.

The p73 protein, a homologue of the tumour-suppressor protein p53, can activate p53-responsive promoters and induce apoptosis in p53-deficient cells. Here we report that some tumour-derived p53 mutants can bind to and inactivate p73. The binding of such mutants is influenced by whether TP53 (encoding p53) codon 72, by virtue of a common polymorphism in the human population, encodes Arg or Pro. The ability of mutant p53 to bind p73, neutralize p73-induced apoptosis and transform cells in cooperation with EJ-Ras was enhanced when codon 72 encoded Arg. We found that the Arg-containing allele was preferentially mutated and retained in squamous cell tumours arising in Arg/Pro germline heterozygotes. Thus, inactivation of p53 family members may contribute to the biological properties of a subset of p53 mutants, and a polymorphic residue within p53 affects mutant behaviour.

Syphilis is a contagious sexually transmitted infection notable for its complex array of systemic presentations. It is caused by the spirochaete Treponema pallidum and although once considered to be a largely historical condition in the UK, the recent rise in incidence makes syphilis increasingly relevant when considering unusual presentations in at-risk patients. The disease has three stages: primary, secondary and tertiary. The tertiary stage is associated with a plethora of neurological features ranging from psychosis to seizure caused by direct invasion of the spirochaete into the central nervous system. Here we describe the case of a 45-year-old man presenting with tonic clonic seizures on a background of balance and visual problems. Following normal examination and routine investigations further serology confirmed a diagnosis of neurosyphilis. The patient was started on appropriate treatment and made an excellent clinical recovery.